Insights into metabolic changes during epidermal differentiation as revealed by multiphoton microscopy with fluorescence lifetime imaging.

Malak M, Qian C, James J, Nair S, Grantham J, Ericson MB

Sci Rep 15 (1) 6377 [2025-02-21; online 2025-02-21]

Rapid developments in the field of organotypic cultures have generated a growing need for effective and non-invasive methods for quality control during tissue development. In this study, we correlate metabolic changes with epidermal differentiation and demonstrate that multiphoton microscopy with fluorescence lifetime imaging (MPM-FLIM) can be applied to monitor epidermal differentiation of keratinocytes with respect to proliferative and differentiated states. In a 2D keratinocyte tissue culture model, increased expression of differentiation markers keratin-1 and keratin-10 was induced with calcium supplementation. An accompanying shift from glycolysis to mitochondrial respiration was detected in metabolic flux assays. Analysis of MPM-FLIM images acquired at 750 nm and 900 nm excitation revealed a decreased relative fraction of intracellular NADH and FAD after high calcium treatment, consistent with increased oxidative phosphorylation. Epidermal differentiation could be monitored over a 96 h period. Discrimination analysis based on k-means clustering generated clusters that correlated well with the duration of high Ca2+ treatment, suggesting that MPM-FLIM can provide useful parameters for monitoring keratinocyte differentiation.

Integrated Microscopy Technologies Gothenburg [Service]

PubMed 39984626

DOI 10.1038/s41598-025-90101-4

Crossref 10.1038/s41598-025-90101-4

pmc: PMC11845624
pii: 10.1038/s41598-025-90101-4